Transfection and Luciferase Assay for Smo Knockout Fibroblasts

Maintenance and transfection of 4C20 Smo^−/− mouse embryonic fibroblasts as well as GLI-luciferase reporter assays were performed as previously described^{11 (link)}. In brief, 4C20 Smo^−/− cells (authenticated by presence of genomic knockout by PCR and absence of SMO protein by western blot and peri-odically tested for mycoplasma contamination; from the P. A. Beachy laboratory) were seeded into 24-well plates and transfected with various plasmids along with a mixture of 8×GLI-luciferase and SV40-Renilla plasmids using TransIT 2020 transfection reagents (Mirus), following manufacturer’s instructions. For each well, 2.5 ng of Smo cDNA, 125 ng 8×GLI-luciferase plasmid, 5 ng SV40-Renilla plasmid and 120 ng GFP expression plasmid were used for transfection. After the cells grew to confluency, the medium was replaced with DMEM containing 0.5% serum and various drugs or vehicle control. Cholesterol–MβCD inclusion complexes were prepared as previously described^{53 (link)}. In brief, a 1:10 molar ratio of cholesterol:MβCD was prepared by adding 9% (w/v) MβCD (Sigma 332615, lot no. STBC2412V, 1.6–2.0 mol CH₃ per unit anhydroglucose) to dried cholesterol, heating to 80 °C and sonicating until a clear solution was achieved. Luciferase activity was measured after 48 h of drug treatment using the Dual luciferase assay kit (Promega) on a Berthold Centro XS3 luminometer.